Genetic linkage, together with techniques for mutational screening of candidate genes, have enabled identification of causative dominant mutations in the genes encoding rhodopsin and peripherin. Globally, about 100 rhodopsin mutations have been found in patients with RP or congenital stationary night blindness. Similarly, approximately 40 mutations have been characterised in the peripherin gene in patients with RP or macular dystrophies (Ott et al. 1990; McWilliam et al. 1989; Dryja et al. 1990; Farrar et al. 1991a,b; Kajiwara et al. 1991; Humphries et al. 1992; Van Soest et al. 1994; Mansergh et al. 1995). Knowledge of the molecular etiology of these retinopathies has stimulated the generation of animal models and the exploration of methods of therapeutic intervention (Farrar et al. 1995; Humphries et al. 1997; Millington-Ward et al. 1997, 1999, 2002; O'Neill et al. 2000).
Osteogenesis imperfecta (OI) is an autosomal dominantly inherited human disease whose molecular pathogenesis is also extremely genetically heterogeneous. OI is often referred to as ‘brittle bone disease’ although additional symptoms such as hearing loss, growth deficiency, bruising, loose joints, blue sclerae and dentinogenesis imperfecta are frequently observed (www.ncbi.nlm.nih.gov/omim). Mutations in the genes encoding the two type I collagen chains (collagen 1A1 and 1A2) comprising the type I collagen heterodimer have been implicated in OI. Indeed, hundreds of dominantly acting mutations in these two genes have been identified in OI patients. Many collagen IA1 and IA2 gene mutations are single point mutations, although a number of insertion and deletion mutations have been found (Willing et al. 1993; Zhuang et al. 1996). Mutations in these genes have also been implicated in Ehlers-Danlos and Marfan syndromes (Phillips et al. 1990; D'Alessio et al. 1991; Vasan N S et al. 1991).
Gene therapies utilizing viral and non-viral delivery systems have been used to treat or study inherited disorders, cancers and infectious diseases. However, many therapies and studies have focused on recessively inherited disorders, the rationale being that introduction and expression of the wild type gene may be sufficient to prevent or ameliorate the disease phenotype. In contrast, gene therapy for dominant disorders such as RP or OI, for example, requires suppression of the dominant disease allele. In addition, there are many polygenic disorders due to co-inheritance of a number of genetic components that together give rise to the disease state. Gene therapies for dominant or polygenic diseases may target the primary defect and require suppression of the disease allele while in many cases still maintaining the function of the normal allele. This is particularly relevant where disease pathology is due to a gain of function mutation rather than to reduced levels of wild type protein. Alternatively, suppression therapies may target secondary effects associated with the disease pathology such as programmed cell death or apoptosis, which has been observed in many inherited disorders.
Suppression effectors have been used previously to achieve specific suppression of gene expression. Modifications have been made to oligonucleotides (e.g., phosphorothioates) to increase resistance to nuclease degradation, binding affinity and uptake (Cazenave et al. 1989; Sun et al. 1989; McKay et al. 1996; Wei et al. 1996). In some instances, antisense and ribozyme suppression strategies have led to the reversal of a tumor phenotype by reducing expression of a gene product or by cleaving a mutant transcript at the site of the mutation (Carter and Lemoine 1993; Lange et al. 1993; Valera et al. 1994; Dosaka-Akita et al. 1995; Feng et al. 1995; Quattrone et al. 1995; Ohta et al. 1996; Lewin et al. 1998). For example, neoplastic reversion was obtained using a ribozyme targeted to an H-ras mutation in bladder carcinoma cells (Feng et al. 1995). Ribozymes have also been proposed as a means of both inhibiting gene expression of a mutant gene and of correcting the mutant by targeted trans-splicing (Sullenger and Cech 1994; Jones et al. 1996). Ribozymes can be designed to elicit autocatalytic cleavage of RNA targets, however, the inhibitory effect of some ribozymes may be due in part to an antisense effect due to the antisense sequences flanking the catalytic core which specify the target site (Ellis and Rodgers 1993; Jankowsky and Schwenzer 1996). Ribozyme activity may be augmented by the use of, for example, non-specific nucleic acid binding proteins or facilitator oligonucleotides (Herschlag et al. 1994; Jankowsky and Schwenzer 1996). Multitarget ribozymes (connected or shotgun) have been suggested as a means of improving efficiency of ribozymes for gene suppression (Ohkawa et al. 1993).
Triple helix approaches have also been investigated for sequence specific gene suppression. Triplex forming oligonucleotides have been found in some cases to bind in a sequence specific manner (Postel et al. 1991; Duval-Valentin et al. 1992; Hardenbol and Van Dyke 1996; Porumb et al. 1996). Similarly, peptide nucleic acids have been shown to inhibit gene expression (Hanvey et al. 1991; Knudson and Nielsen 1996; Taylor et al. 1997). Minor groove binding polyamides can bind in a sequence specific manner to DNA targets and hence may represent useful small molecules for future suppression at the DNA level (Trauger et al. 1996). In addition, suppression has been obtained by interference at the protein level using dominant negative mutant peptides and antibodies (Herskowitz 1987; Rimsky et al. 1989; Wright et al. 1989). In some cases suppression strategies have lead to a reduction in RNA levels without a concomitant reduction in proteins, whereas in others, reductions in RNA have been mirrored by reductions in protein.
A new tool for modulating or suppressing gene expression has also been described called RNA interference (RNAi) or small interfering RNA (siRNA) or double stranded RNA (dsRNA) (Fire, 1998). The silencing effect of complementary double stranded RNA was first observed in 1990 in petunias by Richard Joergensen and termed cosuppression (Jorgensen, 1996). RNA silencing was subsequently identified in C. elegans by Andrew Fire and colleagues (Fire, 1998) who coined the term RNA interference (RNAi). The applications for this biological tool have now been extended to many species as RNAi has been shown to be effective in both mammalian cells and animals (Caplen, 2001; Elbashir, 2001; Yang, 2001; Paddison, 2002; Krichevsky, 2002; Lewis, 2002; Miller et al. 2003). An important feature of dsRNA or siRNA or RNAi is the double stranded nature of the RNA and the absence of large overhanging pieces of single stranded RNA, although dsRNA with small overhangs and with intervening loops of RNA has been shown to effect suppression of a target gene.
The pathway for silencing gene expression involving long (>30 nucleotides) double stranded RNA molecules has been elucidated and is thought to work via the following steps (shown in Drosophila melanogaster) (Zamore, 2001). Firstly, the long dsRNA is cleaved into siRNA approximately 21 nucleotides in length. This siRNA targets complimentary mRNA sequence, which is degraded. However, in mammals it has been found that long dsRNA triggers a non-specific response causing a decrease in all mRNA levels. This general suppression of protein synthesis is mediated by a dsRNA dependent protein kinase (PKR) (Clemens, 1997). Elbashir et al. were able to specifically suppress target mRNA with 21 nucleotide siRNA duplexes. Notably, siRNA bypassed the non-specific pathway and allowed for gene-specific inhibition of expression (Elbashir, 2001; Caplen, 2001). dsRNA can be delivered as synthesized RNA and or by using a vector to provide a supply of endogenously generated dsRNA. dsRNA may be locally or systemically delivered (Lewis, 2002; Miyagishhi, 2002; Paul, 2002; Siu, 2002). Indeed functional siRNAs have been generated both in cells and in transgenic animals and have been delivered using a variety of vector systems including lentivirus (McCaffrey et al. 2003, McManus et al. 2003, Sharp et al. 2003).
Strategies for differentiating between normal and disease alleles and switching off the disease allele using suppression effectors that target the disease mutation are problematic because frequently disease and normal alleles differ by only a single nucleotide. For example, a hammerhead ribozyme that cleaves only at an NUX site is not effective for targeting all point mutations. A further difficulty inhibiting development of gene therapies is the heterogeneous nature of some dominant disorders—many different mutations in the same gene give rise to a similar disease phenotype. Indeed, certain mutations may occur in only one patient. Development of specific gene therapies for each of these mutations may be prohibitive in terms of cost. Examples in which multiple genes and/or multiple mutations within a gene can give rise to a similar disease phenotype include OI, familial hypercholesteremia, and RP. Disease mutations are often single nucleotide changes. As a result differentiating between the disease and normal alleles may be difficult. Some suppression effectors require specific sequence targets, for example, hammerhead ribozymes cleave at NUX sites and hence may not be able to target many mutations. Notably, the wide spectrum of mutations observed in many diseases adds additional complexity to the development of therapeutic strategies for such disorders—some mutations may occur only once in a single patient. A further problem associated with suppression is the high level of homology present in coding sequences between members of some gene families. This can limit the range of target sites for suppression which will enable specific suppression of a single member of such a gene family. A need therefore exists for compositions and methods for suppressing gene expression while also providing for the expression of a non-disease causing allele of the gene that avoids recognition by the suppression effector.